Ligand-induced Desensitization of the Human CXC Chemokine Receptor-2 Is Modulated by Multiple Serine Residues in the Carboxyl-terminal Domain of the Receptor

doi:10.1074/jbc.272.13.8207

Ligand-induced Desensitization of the Human CXC Chemokine Receptor-2 Is Modulated by Multiple Serine Residues in the Carboxyl-terminal Domain of the Receptor*

From the ^‡ Department of Cell Biology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-2175, the
^¶ Veterans Affairs Medical Center, Nashville, Tennessee 37212-2637, and
^§ SmithKline Beecham Pharmaceuticals, Department of Immunology, King of Prussia, Pennsylvania 19406-0939

^∥ To whom correspondence should be addressed. Tel.: 615-343-7777; Fax: 615-343-4539.

Abstract

We have characterized the ligand-enhanced phosphorylation of the CXC chemokine receptor-2 (CXCR2) in a series of clonal 3ASubE cell lines expressing receptors truncated or mutated in the carboxyl-terminal domain. Truncation of CXCR2 by substitution of a stop codon for Ser-342 (342T) or Ser-331 (331T) results in total loss of melanoma growth stimulatory activity/growth-related protein (MGSA/GRO)-enhanced receptor phosphorylation, which cannot be explained based upon altered ligand binding affinity or receptor number. 3ASubE cells expressing 342T or CXCR2 with mutation of Ser-342, −346, −347, and −348 to alanine (4A) exhibit strong mobilization of Ca²⁺ in response to ligand (interleukin-8 or MGSA/GRO), with a recovery phase significantly slower than that of cells expressing wild type (WT) CXCR2. In contrast to the WT CXCR2, which is 93% desensitized by 20 nM ligand, the 331T, 342T, and 4A CXCR2 mutants do not undergo significant ligand-induced desensitization, and respond to a second ligand challenge by mobilizing Ca²⁺. The 3ASubE cells expressing CXCR2 with mutation of Ser-346, −347, and −348 to alanine, or with mutation of only one serine in this domain, continue to be phosphorylated in response to ligand and are 60–70% desensitized following the initial ligand challenge. WT CXCR2 phosphorylation and desensitization occur in <1 min, while receptor sequestration is a much later event (30-60 min). However, mutant receptors that are neither phosphorylated nor desensitized in response to ligand are <10% sequestered 60 min following ligand challenge. These data demonstrate for the first time that ligand binding to CXCR2 results in receptor phosphorylation, desensitization, and sequestration and that serine residues 342 and 346–348 participate in the desensitization and sequestration processes.

Footnotes

↵* This research was supported by National Institutes of Health Grants CA34590 and 5P30 AR41943, a Department of Veterans Affairs Merit and Associate Career Scientist Award (to A. R.), and a grant from the Medical Research Council of Canada (to S. M.). The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

CXC

chemokine, chemokine with the first two amino acids separated by an intervening amino acid

3A

CXCR2 mutant with serine to alanine substitutions at positions 346, 347, and 348

4A

CXCR2 mutant with serine to alanine substitutions at positions 342, 346, 347, and 348

342T

CXCR2 truncated at the Ser-342 by placing a stop codon at Ser-342

331T

CXCR2 truncated at Ser-331 by placement of a stop codon in place of Ser

352T

CXCR2 truncated at Ser-352 by placing a stop codon at Ser-352

293 cells

human embryonic kidney cell line

293T₂

human embryonic kidney 293 cells expressing transfected human CXCR2

CC

chemokines, chemokines with the first two cysteines positioned side-by-side

CMV

cytomegalovirus

CXCR1

receptor for CXC chemokines formerly referred to as IL-8 receptor A

CXCR2

receptor for CXC chemokines formerly defined as IL-8 receptor B

C5a

complement fragment 5a

DMEM

Dulbecco's modified Eagle's medium

FBS

fetal bovine serum

fMLP

N-formyl-methionyl-leucyl-phenylalanine

FURA-2

fluorescence indicator for free calcium

IL-8

interleukin-8

MGSA/GRO

melanoma growth-stimulatory activity/growth-related protein

PBS

phosphate-buffered saline

TPA

12-O-tetradecanoylphorbol-13-acetate

WT

wild type.
- Received April 30, 1996.
- Revision received January 21, 1997.